Natural extracts for modulating pp2a methylation, and providing antioxidant and anti inflammatory activity

ABSTRACT

Compositions containing natural and botanical extracts for use in inhibiting one, two, or three of (a)-(c): (a) demethylation of PP2A by PME-1 methylesterase; (b) formation of free radicals and reactive oxygen species; and/or (c) inflammation. These compositions include an extract of one or more botanicals selected from the group consisting of: juniper berry fruit, schisandra fruit, strawberry fruit, avocado seeds, black raspberry seeds, blueberry seeds, celery seeds, cranberry seeds, fennel seeds, grape seeds, guarana seeds, red raspberry seeds, maca root, goldenseal root, turmeric root, magnolia bark, pygeum bark, red raspberry leaf, almond, cocoa powder, Echinacea angustifolia, prickly pear cactus and walnut.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/027,607, filed Jul. 22, 2014, and herein incorporated by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to compositions containing natural andbotanical extracts for use in inhibiting demethylation of PP2A by PME-1methylesterase; formation of free radicals and reactive oxygen species;and/or inflammation.

BACKGROUND

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, hasbeen implicated in a broad range of cellular functions anywhere fromdevelopment to disease. Consisting of a scaffolding (A), regulatory (B)and catalytic (C) subunit, as shown in FIG. 1, this trimeric holoenzymeis highly regulated through structural assembly, post-translationalmodifications and small molecule interactions. Methylation of PP2A'scarboxy-terminal tail has not only been implicated in modulating itsactivity and specificity but it has also been shown to be of particularimportance in neurodegenerative diseases such as Alzheimer's andParkinson's disease. For this reason, modulators of PP2A's methylationstate are of particular importance.

Protein phosphatase 2A (PP2A) associates with a variety of regulatorysubunits. (Janssens, V., Gloris, J., Biochem. J. 353 (Pt. 3): 417-39(2001)). The predominant form in neuronal tissue is a trimer composed ofa dimeric core composed of a 65 kilodalton (kDa) A subunit and the 36kDa PP2A catalytic C subunit associated with one of several differentregulatory B subunits. Whereas the A and C subunits are present more orless uniformly, the B subunit is variable and confers substratespecificity and subcellular localization to each PP2A holoenzyme trimer.The number and types of B subunits present is subject to developmentalregulation and is cell type specific.

The variable B subunits of PP2A are classified into four families: (1)the B family with four isoforms (α, β, γ, δ); (2) the B′ family withfive isoforms (α, β, γ, δ, ε); (3) the B″ family; and (4) the B′″family. The PP2A ABαC heterotrimer appears to be the major phosphatasein brain responsible for dephosphorylation of tau. (Kamibayashi, C. etal., J. Biol. Chem. 269 (31): 20139-148 (1994); Sontag, E., et al., J.Neuropathol. Exp. Neurol. 63 (4): 287-301 (2004)).

The alpha-carboxyl of the C terminal leucine residue of the catalyticsubunit of PP2A is subject to methyl esterification and methyl-esterhydrolysis, and the methylation state of PP2A regulates heterotrimerformation. (Tokstykh, T. et al., EMBO J. 19 (21): 5682-91 (2000); Wu, J.et al., EMBO J. 19 (21): 5672-81 (2000); Wei, H. et al., J. Biol. Chem.276 (2): 1570-77 (2001); Yu, X X, et al., Mol. Biol. Cell 12 (1): 185-99(2001)). Two enzymes are involved in controlling the methylation stateof PP2A: (1) an S-adenosylmethionine-dependent PP2A-specific proteinmethyltransferase (“PPMT”), which adds the methyl group and (2) aPP2A-specific protein methylesterase (“PPME”), which removes the methylgroup. PP2A methylation promotes PP2A ABαC trimer assembly. Anydeficiency in methylation is expected to preclude PP2A ABαC heterotrimerformation, thereby leading to a deficiency in tau dephosphorylation, tauhyperphosphorylation and the formation of neurofibrillary tangles.(Vafai, S. B., Stock, J. B., FEBS Lett. 518 (1-3): 1-4 (2002)).

Homocysteine, a sulfur-containing amino acid that can be eitherremethylated to methionine or undergo a trans-sulfuration reaction tocystathionine, plays a key role in methylation metabolism (see FIG. 1).The conversion of homocysteine to methionine occurs in all tissues.Methionine is activated by ATP in the presence of methionine adenosyltransferase to form the methyl donor, S-adenosylmethionine (“SAM”).SAM-dependent methylation reactions in the presence of SAM-dependentmethyltransferases result in the formation of S-adenosylhomocysteine(“SAH”), which is cleaved by SAH hydrolase to form adenosine andhomocysteine. This reaction is reversible with the equilibrium favoringthe condensation of homocysteine and adenosine. Under normal conditions,homocysteine is rapidly methylated, which favors the further cleavage ofSAH. Homocysteine accumulation leads to global decreases in cellularmethylation by the condensation of homocysteine with adenosine to formSAH, which acts as a product inhibitor in cellular methylationreactions. In the United States, 5-10% of the general population haselevated plasma homocysteine, and this imbalance increases to 30-40% inof the elderly. (Selub J., et al., Ann. Intern. Med. 131 (5): 331-39(1999)). See Vafai, S. B., Stock, J. B., FEBS Lett. 2: 518 (2002).

Over the last several years, data has emerged in clinical literaturesuggesting a direct association between elevated plasma homocysteine andthe occurrence of AD. Seshadri et al., (N Engl J Med 346 (7): 476-83(2002)), demonstrated that elevated homocysteine is a risk factor forAD. After adjusting for other AD risk factors, the study concluded thatplasma homocysteine levels greater than 14 μM coincided with about a2-fold increased risk for developing AD with an additional 40% increasedrisk with each 5 μM incremental rise. Other diseases, conditions ordisorders associated with elevated plasma homocysteine include, but arenot limited to, atherosclerosis; neurodegenerative disorders, such asParkinson's disease; cerebrovascular disorders (i.e., disorderspertaining to blood vessels in the brain), such as stroke;neuropsychiatric disorders, such as bipolar disorder and schizophrenia;diabetes (type II), and arthritis.

An analysis of the clinical and basic science literature indicates thata methylation defect resulting from elevated homocysteine could lead tolowered levels of PP2A methylation that would result in lowered PP2AABαC, which is believed to lead to tau hyperphosphorylation,neurofibrillary tangle formation, and dementia (Vafai and Stock, FEBSLett 518(1-3): 1-4 (2002)).

Cellular pathways for removing plasma homocysteine require folate,Vitamin B₆ and B₁₂, and thus high homocysteine levels are expected inmice fed diets deficient in these components. This was demonstratedusing, male C57BL/J6 mice. One set of 4 week old mice were placed on adiet that contained folate, vitamin B₆, and vitamin B₁₂ and another setwere fed diets that lacked these vitamins. The mice were allowed freeaccess to both food and water. After nine weeks on their respectivediets, each mouse was sacrificed by cervical dislocation. Blood sampleswere collected for measurement of plasma homocysteine and the brain wasremoved and quickly frozen in liquid nitrogen for further analysis oftau phosphorylation. As expected the vitamin-deficient diets causedsubstantial increases in plasma Hcy and brain SAH. These increases wereaccompanied by elevated levels of Tau phosphorylation. CP13 and PHF1 aremonoclonal antibodies that are specific for phosphorylated tau epitopes.TG5 is a monoclonal antibody that recognizes tau independent of itsstate of phosphorylation; it thereby provides a control showing thattotal levels of tau expression are unaffected by diet. Mice raised ondiets deficient in folate, B₁₂, and B₆ had dramatically elevated levelsof total plasma homocysteine, brain S-adenosyl homocysteine and elevatedlevels of tau phosphorylation. S-Adenosyl methionine levels were notsignificantly affected.

The demographics of aging in the United States population, combined witha lack of effective treatments, have heightened the need for ADtherapies. Moreover, the development of preventives would be an evengreater contribution to public health. A protective agent that could betaken over many years to reduce the risk of AD or to substantively delayits onset would be an invaluable breakthrough.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention provides compositions containingnatural and botanical extracts for use in inhibiting one, two, or threeof (a)-(c): (a) demethylation of PP2A by PME-1 methylesterase; (b)formation of free radicals and reactive oxygen species; and/or (c)inflammation. These compositions include an extract of one or morebotanicals selected from the group consisting of: juniper berry fruit,schisandra fruit, strawberry fruit, avocado seeds, black raspberryseeds, blueberry seeds, celery seeds, cranberry seeds, fennel seeds,grape seeds, guarana seeds, red raspberry seeds, maca root, goldensealroot, turmeric root, magnolia bark, pygeum bark, red raspberry leaf,almond, cocoa powder, Echinacea angustifolia, prickly pear cactus andwalnut.

In one particularly preferred embodiment, the compositions contain acombination of extracts that inhibit all three of (a) demethylation ofPP2A by PME-1 methylesterase; (b) formation of free radicals andreactive oxygen species; and (c) inflammation. The combination ofextracts can be combined in a single composition.

Another aspect of the present invention provides a method of preparing abotanical extract that includes introducing a raw botanical to a solventhaving a dielectric constant ranging from about 2.3 to about 25 andallowing the mixture to reside for a sufficient time to obtain a crudeextract. The method further includes filtering out remaining rawbotanical and evaporating the solvent from the crude extract underreduced pressure to obtain a dried crude extract, and washing the driedcrude extract with a polar solvent having a dielectric constant of noless than 30 (e.g., water), heating the resulting mixture, and allowingto cool. The method can further include collecting a filtrate from thecooled mixture mentioned above and washing the collected filtrate with anon-polar solvent having a dielectric constant of no more than 2.0(e.g., cyclohexane, hexane, heptane and isooctane). The method canfurther include filtering and drying the mixture mentioned above toobtain the botanical extract.

Another aspect of the present invention provides a method for inhibitingdemethylation of PP2A by PME-1 methylesterase comprising administeringto a subject in need thereof an effective amount of a botanical extract.Subjects that are in need of inhibition of demethylation of PP2A byPME-1 methylesterase, and hence can be administered the presentlydisclosed extracts or compositions, include, but are not limited to,subjects that exhibit, or are at risk for exhibiting, Tauhyper-phosphorylation, α-synculein hyper-phosphorylation, and/orabnormally elevated homocysteine levels.

In another aspect of the present invention, the subject exhibits, or isat risk for exhibiting, a skin disorder, medical condition or disease.In one embodiment, the subject desires to maintain healthy skin andprevent skin aging.

Another aspect of the present invention provides a method for inhibitinginflammation in a subject comprising administering to a subject aneffective amount of a botanical extract.

Another aspect of the present invention provides a method for inhibitingthe formation of free radicals and reactive oxygen species in a subjectcomprising administering to a subject an effective amount of a botanicalextract obtained from at least one source selected from the groupconsisting of grape seed, guarana seed, red raspberry seed, prickly pearcactus and turmeric root.

Any one of the above-described extracts, and compositions containingthese extracts, can be, in certain embodiments, topically administered,such as, for example, as a cream, lotion, cleanser, ointment or otherpharmaceutically acceptable topical dosage form. Alternatively, any oneof the above-described extracts, and compositions containing theseextracts, can be, in certain embodiments, orally administered, such as,for example, as a pill, tablet, capsule, syrup or a drink or otherpharmaceutically acceptable oral dosage form. In either topical or oralform, the extract can be administered together with other botanicals andvitamins.

The invention is further directed to the general and specificembodiments defined, respectively, by the Numbered Embodiments appendedhereto, which are incorporated by reference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a flow chart of PP2A assembly and methylation.

FIG. 2 presents a bar graph of PP2A-AC phosphatase activity.

DETAILED DESCRIPTION OF THE INVENTION

The invention can be more fully appreciated by reference to thefollowing description, including the examples. Unless otherwise defined,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described herein. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

For the sake of brevity, all publications, including patentapplications, patents, and other citations mentioned herein, areincorporated by reference in their entirety. Citation of any suchpublication, however, shall not be construed as an admission that it isprior art to the present invention.

Terms and Definitions

As used herein, the term “about” or “approximately” means within anacceptable range for a particular value as determined by one skilled inthe art, and may depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system ortechnique. For example, “about” can mean a range of up to 20%, up to10%, up to 5%, or up to 1% or less on either side of a given value.Alternatively, with respect to biological systems or processes, the term“about” can mean within an order of magnitude, within 5 fold, or within2 fold on either side of a value. Numerical quantities given herein areapproximate unless stated otherwise, meaning that the term “about” or“approximately” can be inferred when not expressly stated.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about.” It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to both the actual givenvalue and the approximation of such given value that would reasonably beinferred based on the ordinary skill in the art, including equivalentsand approximations due to the experimental and/or measurement conditionsfor such given value. Whenever a yield is given as a percentage, suchyield refers to a mass of the entity for which the yield is given withrespect to the maximum amount of the same entity for which that could beobtained under the particular stoichiometric conditions. Concentrationsthat are given as percentages refer to mass ratios, unless indicateddifferently.

As used herein, the terms “a,” “an,” and “the” are to be understood asmeaning both singular and plural, unless explicitly stated otherwise.Thus, “a,” “an,” and “the” (and grammatical variations thereof whereappropriate) refer to one or more.

A group of items linked with the conjunction “and” should not be read asrequiring that each and every one of those items be present in thegrouping, but rather should be read as “and/or” unless expressly statedotherwise. Similarly, a group of items linked with the conjunction “or”should not be read as requiring mutual exclusivity among that group, butrather should also be read as “and/or” unless expressly statedotherwise. Furthermore, although items, elements or components of theinvention may be described or claimed in the singular, the plural iscontemplated to be within the scope thereof, unless limitation to thesingular is explicitly stated.

The terms “comprising” and “including” are used herein in their open,non-limiting sense. Other terms and phrases used in this document, andvariations thereof, unless otherwise expressly stated, should beconstrued as open ended, as opposed to limiting. Thus, the term“example” is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof. Similarly,adjectives such as “conventional,” “traditional,” “normal,” “criterion,”“known,” and terms of similar meaning should not be construed aslimiting the item described to a given time period or to an itemavailable as of a given time, but they should be read to encompassconventional, traditional, normal, or criterion technologies that may beavailable or known now or at any time in the future. Likewise, wherethis document refers to technologies that would be apparent or known toone of ordinary skill in the art, such technologies encompass thoseapparent or known to the skilled artisan now or at any time in thefuture.

The presence of broadening words and phrases such as “one or more,” “atleast,” “but not limited to” or other like phrases in some instancesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent. As will becomeapparent to one of ordinary skill in the art after reading thisdocument, the illustrated embodiments and their various alternatives maybe implemented without confinement to the illustrated examples.

The term “composition” is intended to encompass a product including theherein described extracts and the inert ingredient(s) (pharmaceuticallyacceptable excipients) that make up the carrier, as well as any productwhich results, directly or indirectly, from combination, complexation,or aggregation of any two or more of the ingredients, or fromdissociation of one or more of the ingredients, or from other types ofreactions or interactions of one or more of the ingredients. In certainembodiments, a “composition,” as used herein, is pharmaceuticallyacceptable and suitable for oral administration. In alternativeembodiments, a “composition,” as used herein, is pharmaceuticallyacceptable and suitable for topical administration.

The term “carrier” refers to an adjuvant, vehicle, or excipients, withwhich the compound is administered. In certain embodiments of thisinvention, the carrier is a solid carrier. Suitable pharmaceuticalcarriers include those described in Remington: The Science and Practiceof Pharmacy, 21^(st) Ed., Lippincott Williams & Wilkins (2005).

The term “dosage form,” as used herein, is the form in which the dose isto be administered to the subject or patient. The active extract isgenerally administered as part of a formulation that includes nonmedicalagents. The dosage form has unique physical and pharmaceuticalcharacteristics. Dosage forms, for example, can be solid, liquid, gel orgaseous. “Dosage forms” may include for example, a capsule, tablet,caplet, gel caplet (gelcap), syrup, a liquid composition, a powder, aconcentrated powder, a concentrated powder admixed with a liquid ortopical gel or lotion, a chewable form, a swallowable form, adissolvable form, an effervescent, a granulated form, a topical form andan oral liquid solution. In a specific embodiment, the dosage form is asolid dosage form, and more specifically, comprises a tablet or capsule.In another specific embodiment, the dosage form is a topical dosageform, and more specifically, comprises a gel, lotion or other formsuitable for application to human skin.

The term “pharmaceutically acceptable,” as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to ananimal (e.g., human) according to their intended mode of administration(e.g., oral or topical).

A “pharmaceutically acceptable excipient” refers to a substance that isnon-toxic, biologically tolerable, and otherwise biologically suitablefor administration to a subject, such as an inert substance, added to apharmacological composition or otherwise used as a vehicle, carrier, ordiluents to facilitate administration of an agent and that is compatibletherewith. Examples of excipients include calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils, and polyethylene glycols. Suitablepharmaceutical carriers include those described in Remington: TheScience and Practice of Pharmacy, 21^(st) Ed., Lippincott Williams &Wilkins (2005).

As used herein, the term “inert” refer to any inactive ingredient of adescribed composition. The definition of “inactive ingredient” as usedherein follows that of the U.S. Food and Drug Administration, as definedin 21 C.F.R. 201.3(b)(8), which is any component of a drug product otherthan the active ingredient.

As used herein, “suitable for oral administration” or “suitable fortopical administration” refers to a sterile, pharmaceutical productproduced under good manufacturing practices (GMP). The term “suitablefor oral administration” or “suitable for topical administration” can,when specified, also mean approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals (e.g. mammals), andmore particularly in humans.

As used herein, the term “raw botanical” as used herein refers to afresh or processed (e.g. cleaned, frozen, dried, sliced, dissolved, orliquefied) part of a single species of plant or natural material, or afresh or processed alga or macroscopic fungus. Raw botanicals can becommercially obtained from, for example, Prickly Pear Products, LLC,Mesa, Ariz., Starwest Botanicals, Sacramento, Calif.,www.HerbStoreUSA.com, Frontier Natural Products CO-OP, andwww.znaturalfoods.com.

As used herein, the term “disorder” is used interchangeably with“disease” or “condition”. For example, a neurological disorder alsomeans a neurological disease or a neurological condition.

The terms “treat,” “treating,” and “treatment” cover therapeutic methodsdirected to a disease-state in a subject and include: (i) preventing thedisease-state from occurring, in particular, when the subject ispredisposed to the disease-state but has not yet been diagnosed ashaving it; (ii) inhibiting the disease-state, e.g., arresting itsdevelopment (progression) or delaying its onset; and (iii) relieving thedisease-state, e.g., causing regression of the disease state until adesired endpoint is reached. These terms also include ameliorating asymptom of a disease (e.g., reducing the pain, discomfort, or deficit),wherein such amelioration may be directly affecting the disease (e.g.,affecting the disease's cause, transmission, or expression) or notdirectly affecting the disease.

As used in the present disclosure, the term “effective amount” isinterchangeable with “therapeutically effective amount” and means anamount or dose of a compound or composition effective in treating theparticular disease, condition, or disorder disclosed herein, and thus“treating” includes producing a desired preventative, inhibitory,relieving, or ameliorative effect. In methods of treatment according tothe invention, “an effective amount” of at least one compound isadministered to a subject (e.g., a mammal). The “effective amount” willvary, depending on the compound, the disease (and its severity), thetreatment desired, age and weight of the subject, etc.

As used herein, the phrase “in combination” refers to agents that aresimultaneously administered to a subject. It will be appreciated thattwo or more agents are considered to be administered “in combination”whenever a subject is simultaneously exposed to both (or more) of theagents. Each of the two or more agents may be administered according toa different schedule; it is not required that individual doses ofdifferent agents be administered at the same time, or in the samecomposition. Rather, so long as both (or more) agents remain in thesubject's body, they are considered to be administered “in combination”.

As used herein, the term “modulate” refers to change in a parameter(e.g., a change in a binding interaction or an activity, etc.).Modulation can refer to an increase or a decrease in the parameter(e.g., an increase or decrease in binding, an increase or decrease inactivity, etc.).

As used herein, the term “modulator” refers to an agent that alterslevel and/or activity of its target (e.g., in the GPCR signaltransduction pathway). In some embodiments, a modulator altersinteraction between a protein in the GPCR signal transduction pathwayand one or more other entities. In some embodiments, a modulator altersinteraction between a modulator alters interaction between a protein inthe GPCR signal transduction pathway and a substrate. Determination ofwhether an agent is a modulator can be performed directly or indirectly.Determination of whether an agent modulates an interaction can beperformed directly, e.g., using an assay that detects the interactionbetween a protein in the GPCR signal transduction pathway and asubstrate. Determination of whether an agent modulates an interactioncan be performed with a technique that indirectly detects modulation,e.g., a technique that detects a biological activity that is downstreamof, and dependent on, the protein-substrate interaction.

As used herein, the term “skin irritant” refers to a extract that, whenapplied to skin or a skin equivalents, elicits a cellular responsecharacterized by the expression of an “irritant responsive gene.”Examples of known skin irritants include, but are not limited to, sodiumdodecyl sulfate (“SDS”), calcipotriol, and trans-retinoic acid. The term“skin irritant” is also intended to encompass unknown or suspectedirritants, including but not limited to, those containing in somepharmaceuticals, cosmetics, and consumer products. In embodiments of thepresent invention, the presently disclosed compositions are free of, orsubstantially free of, skin irritants.

The terms “individual,” “subject,” and “patient” are usedinterchangeably herein and can be a vertebrate, in particular, a mammal,more particularly, a primate (including non-human primates and humans)and include a laboratory animal in the context of a clinical trial orscreening or activity experiment. Thus, as can be readily understood byone of ordinary skill in the art, the compositions and methods of thepresent invention are particularly suited to administration to anyvertebrate, particularly a mammal, and more particularly, a human.

As used herein, the term “cognitive function” refers to the ability toperform mental tasks, such as thinking, learning, judging, remembering,computing, controlling motor functions, and the like. The expression“resilience of cognitive function” refers to the ability of functionalelements of cognitive function to resist deterioration over time. Asused herein, the term “cognitive function enhancing amount” refers tothat amount of the composition of the present invention that willnoticeably impact the ability to perform mental tasks, as measured bytests for memory, computation, attention, or other mental or cognitiveattribute, or as suggested by an individual's perception of his or herabilities in these realms.

As used herein, the term “G-protein mediated condition” refers anydisease or other deleterious condition for which the appearance,incidence, and/or severity of one or more symptoms correlates withchanges in a G-protein signaling cascade. In some embodiments, one ormore symptoms of the disease or condition is caused by a defect oralteration in G-protein signaling.

As used herein, the term “comestible” refers to a material that issuitable for human consumption, including a material that can beingested by oral and by a non-oral means, e.g., an inhalant or a snuff.For purposes of the present invention, the term includes supplemented orenhanced foods.

The terms “dietary supplement” and “nutritional supplement” are usedinterchangeably herein to mean (1) a product intended to supplement thediet that bears or contains one or more of the following dietaryingredients: [A] a vitamin, [B] a mineral, [C] an herb or otherbotanical, [D] an amino acid, [E] a dietary substance for use by man tosupplement the diet by increasing the total dietary intake; or (F) aconcentrate, metabolite, constituent, extract, or combination of anyingredient described in clause (A), (B), (C), (D), or (E); and (2) aproduct that (A)(i) is intended for ingestion; (B) is not representedfor use as a conventional food or as a sole item of a meal or the diet;and (C) is labeled as a dietary supplement.

The term “health” or “healthy” as used herein refers to a generalcondition of the body or mind with reference to soundness and vigor, aswell as freedom from disease or ailment.

The term “partitioning” as used herein refers to a process that takesadvantage of the differential solubility of a substance in two solvents.

The terms “soluble” and “solubility” refer to the property of beingsusceptible to being dissolved in a specified fluid (solvent). The term“insoluble,” as used herein refers to the property of a material thathas minimal or limited solubility in a specified solvent.

The term “solvent” as used herein refers to a substance, usually liquid,capable of dissolving or dispersing one or more other substances.Chemists have classified solvents into two broad categories according totheir polarity: polar and nonpolar. A common measure of the polarity ofa solvent is the dielectric constant. The term “polar solvent” as usedherein refers to a compound that is composed of polar molecules. A“polar molecule” is one in which there is some separation of charge inthe chemical bonds, so that one part of the molecule has a slightpositive charge and the other a slight negative charge. Polar solventsmay be further classified as protic or aprotic. The term “protic” refersto a hydrogen atom attached to an electronegative atom, while the term“aprotic” refers to a molecule that does not contain an O—H bond. A“polar protic solvent” can be represented by the general formula ROH;the polarity of the polar protic solvent stems from the bond dipole ofthe O—H bond. Examples of polar protic solvents include, but are notlimited to, water, alcohols, and acetic acid. A “dipolar aproticsolvent” is one that contains a bond that has a large bond dipole.Typically, this bond is a multiple bond between carbon and either oxygenor nitrogen. Most dipolar aprotic solvents contain a C—O double bond.Examples of dipolar aprotic solvents include, but are not limited to,acetone and ethyl acetate. As the number of —CH₂— groups in ROHincreases, and the relative amount of hydrocarbon character increases,the polarity decreases. The term “nonpolar solvent” refers to compoundsthat have low dielectric constants and are not miscible with water.Examples of nonpolar solvents include, but are not limited to, benzene,carbon tetrachloride, and hexanes.

The term “well-being” as used herein refers to a subject's physical andmental soundness.

A composition of the present invention, alone or in combination withother active ingredients, may be administered to a subject in a singledose or multiple doses over a period of time, generally by oral ortopical administration. As used herein, the terms “therapeuticallyeffective amount,” “memory-enhancing amount”, and “cognition enhancingamount” refer to the amount of the composition of the invention thatresults in a therapeutic or beneficial effect, including a subject'sperception of health or general well-being, following its administrationto a subject.

It is believed that an increase in the level of PP2A methylation, orPP2A modulation in general, will bring about the protection orenhancement of cognitive functioning, or preventing a cognitive disorderfrom manifesting or deepening. Thus the therapeutic effect of thecompositions of the present invention can exert a protective orenhancing effect on cognitive function; minimize, prevent or amelioratecognitive symptoms of a disease or disorder, or may have any otherbeneficial effect.

The concentration of the substance is selected so as to exert itstherapeutic effect, but low enough to avoid significant side effectswithin the scope and sound judgment of the skilled artisan. Theeffective amount of the composition may vary with the age and physicalcondition of the biological subject being treated, the severity of thecondition, the duration of the treatment, the nature of concurrenttherapy, the specific compound, composition or other active ingredientemployed, the particular carrier utilized, and like factors. Those ofskill in the art can readily evaluate such factors and, based on thisinformation, determine the particular effective concentration of acomposition of the present invention to be used for an intended purpose.

A skilled artisan can determine a therapeutically effective amount ofthe inventive compositions by determining the unit dose. As used herein,a “unit dose” refers to the amount of inventive composition required toproduce a response of 50% of maximal effect (i.e. ED50). The unit dosecan be assessed by extrapolating from dose-response curves derived fromin vitro or animal model test systems. The amount of compounds in thecompositions of the present invention which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. (See, for example, Goodman and Gilman's ThePharmacological Basis of Therapeutics, Joel G. Harman, Lee E. Limbird,Eds.; McGraw Hill, New York, 2001; The Physician's Desk Reference,Medical Economics Company, Inc., Oradell, N.J., 1995; and Drug Facts andComparisons, Facts and Comparisons, Inc., St. Louis, Mo., 1993). Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Various administration patterns will beapparent to those skilled in the art.

The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desiredtherapeutic effect. Preferably, the cognitive function enhancing amountof the compositions of the present invention is administered one or moretimes per day on a regular basis. A typical dose administered to a humanis between about 1 mg and about 10 g of the composition, preferablybetween 1 mg and 1 g of the composition.

Those skilled in the art will recognize that initial indications of theappropriate therapeutic dosage of the compositions of the invention canbe determined in in vitro and in vivo animal model systems, and in humanclinical trials. One of skill in the art would know to use animalstudies and human experience to identify a dosage that can safely beadministered without generating toxicity or other side effects. Foracute treatment where it is desirable to substantially increasemethylated PP2A, it is preferred that the therapeutic dosage be close tothe maximum tolerated dose. For chronic preventive use, lower dosagesmay be desirable because of concerns about long term effects. However,the instant extracts are commonly believed to be safe and have a historyof human use. Alternatively, the composition of the present inventionmay be administered at least once per day in combination with aprescribed drug. For example, the composition of the present inventionmay be administered together with existing anti-cholinesterase drugs nowprescribed for Alzheimer's, with various anti-inflammatory agents, orwith statins.

Reference will now be made to the embodiments of the present invention,examples of which are illustrated by and described in conjunction withthe accompanying examples. While certain embodiments are describedherein, it is understood that the described embodiments are not intendedto limit the scope of the invention. On the contrary, the presentdisclosure is intended to cover alternatives, modifications, andequivalents that can be included within the invention as defined by theappended numbered embodiments.

One aspect of the present invention provides compositions containingnatural and botanical extracts for use in inhibiting one, two, or threeof (a)-(c): (a) demethylation of PP2A by PME-1 methylesterase; (b)formation of free radicals and reactive oxygen species; and/or (c)inflammation. These compositions include an extract of one or morebotanicals selected from the group consisting of: juniper berry fruit,schisandra fruit, strawberry fruit, avocado seeds, black raspberryseeds, blueberry seeds, celery seeds, cranberry seeds, fennel seeds,grape seeds, guarana seeds, red raspberry seeds, maca root, goldensealroot, turmeric root, magnolia bark, pygeum bark, red raspberry leaf,almond, cocoa powder, Echinacea angustifolia, prickly pear cactus andwalnut.

In embodiments in which the compositions inhibit demethylation of PP2Aby PME-1 methylesterase, or that modulate PP2A, the compositions caninclude an extract of one or more of juniper berry fruit, schisandrafruit, strawberry fruit, avocado seeds, black raspberry seeds, blueberryseeds, celery seeds, cranberry seeds, fennel seeds, grape seeds, guaranaseeds, red raspberry seeds, maca root, goldenseal root, turmeric root,magnolia bark, pygeum bark, red raspberry leaf, almond, cocoa powder,Echinacea angustifolia, prickly pear cactus and walnut. In alternativeembodiments in which the compositions inhibit demethylation of PP2A byPME-1 methylesterase, the compositions can include an extract of one ormore of grape seed, guarana seed, red raspberry seed, prickly pearcactus, and turmeric root.

One embodiment of the present invention provides compositions comprisinga methylation modifying compound isolated from a raw botanical material(i) that inhibits at least one enzyme that demethylates PP2A, whereinthe composition inhibits at least 50%, more preferably by at least 90%,of the demethylating activity of the demethylating enzyme as measured bylevels of PP2A methyl esterification; or (ii) that stimulates themethylating activity of at least one enzyme that methylates PP2A. In oneembodiment, the botanical extract contains at least 5 units permicroliter of the activity inhibiting demethylation of the proteinphosphatase 2A (PP2A) enzyme and thereby stimulates methylation of theprotein phosphatase 2A (PP2A) enzyme. In an alternative embodiment, thebotanical extract, or extracts when used in combination, has an IC₅₀value of less than 25 μg/mL, or less than 10 μg/mL, or less than 5μg/mL, as determined according to the procedure of Example 2 herein. Inone embodiment, the presently disclosed compositions modulate the PP2Aenzyme.

In certain embodiments in which the compositions inhibit the formationof free radicals and reactive oxygen species, the compositions caninclude an extract of one or more of grape seed, guarana seed, redraspberry seed, prickly pear cactus, and turmeric root. In oneembodiment, the botanical extract, or extracts when used in combination,has an IC₅₀ value of less than 25 μg/mL, or less than 10 μg/mL, or lessthan 5 μg/mL, as determined according to the procedure of Example 3herein.

In certain embodiments in which the compositions inhibit inflammation(including, e.g., micro-inflammation) the compositions can include anextract of one or more of grape seed, guarana seed, red raspberry seed,prickly pear cactus, and turmeric root. In one embodiment, the botanicalextract, or extracts when used in combination, has an IC₅₀ value of lessthan 25 μg/mL, or less than 10 μg/mL, or less than 5 μg/mL, asdetermined according to the procedure of Example 4 herein.

In certain embodiments, any one of the compositions set forth herein(e.g., compositions containing natural and botanical extracts for use ininhibiting one, two, or three of (a)-(c): (a) demethylation of PP2A byPME-1 methylesterase; (b) formation of free radicals and reactive oxygenspecies; and/or (c) inflammation) includes an extract that is obtainedfrom a solvent having a dielectric constant ranging from about 2.3 toabout 25. For example, the extract can be obtained from one or more ofisopropyl alcohol, toluene, and ethyl acetate. In further embodiments,the composition includes an extract that is substantially free of polarcomponents (e.g., caffeine, sugars, oligosaccharides, etc.) soluble insolvents with a dielectric constant no less than 30 (e.g., water). In astill further embodiment, the extract is substantially free of non-polarcomponents (e.g., lipids, oils) soluble in solvents with a dielectricconstant no more than 2.0 (e.g., cyclohexane, hexane, heptane orisooctane).

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed. In one embodiment, thecomposition includes, or consists essentially of, or consists of, anextract of guarana seed. In one embodiment, the composition includes, orconsists essentially of, or consists of, an extract of red raspberryseed. In one embodiment, the composition includes, or consistsessentially of, or consists of, an extract of prickly pear cactus. Inone embodiment, the composition includes, or consists essentially of, orconsists of, an extract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed and an extract of guarana seed.In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed and an extract of red raspberryseed. In one embodiment, the composition includes, or consistsessentially of, or consists of, an extract of grape seed and an extractof prickly pear cactus. In one embodiment, the composition includes, orconsists essentially of, or consists of, an extract of grape seed and anextract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of guarana seed and an extract of redraspberry seed. In one embodiment, the composition includes, or consistsessentially of, or consists of, an extract of guarana seed and anextract of prickly pear cactus. In one embodiment, the compositionincludes, or consists essentially of, or consists of, an extract ofguarana seed and an extract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of red raspberry seed and an extract ofprickly pear cactus. In one embodiment, the composition includes, orconsists essentially of, or consists of, an extract of red raspberryseed and an extract of turmeric root. In one embodiment, the compositionincludes an extract of prickly pear cactus and an extract of turmericroot.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed, an extract of guarana seed,and an extract of red raspberry seed. In one embodiment, thecompositions includes, or consists essentially of, or consists of, anextract of grape seed, an extract of guarana seed, and an extract ofprickly pear cactus. In one embodiment, the compositions includes, orconsists essentially of, or consists of, an extract of grape seed, anextract of guarana seed, and an extract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed, an extract of red raspberryseed, and an extract of prickly pear cactus. In one embodiment, thecompositions includes, or consists essentially of, or consists of, anextract of grape seed, an extract of red raspberry seed, and an extractof turmeric root. In one embodiment, the compositions includes, orconsists essentially of, or consists of, an extract of grape seed, anextract of prickly pear cactus, and an extract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of guarana seed, an extract of red raspberryseed, and an extract of prickly pear cactus. In one embodiment, thecomposition includes, or consists essentially of, or consists of, anextract of guarana seed, an extract of red raspberry seed, and anextract of turmeric root. In one embodiment, the composition includes,or consists essentially of, or consists of, an extract of guarana seed,an extract of prickly pear cactus, and an extract of turmeric root. Inone embodiment, the compositions include, or consists essentially of, orconsists of, an extract of red raspberry seed, an extract of pricklypear cactus, and an extract of turmeric root.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed, an extract of guarana seed, anextract of red raspberry seed, and an extract of prickly pear cactus. Inone embodiment, the composition includes, or consists essentially of, orconsists of, an extract of grape seed, an extract of guarana seed, anextract of red raspberry seed, and an extract of turmeric root. In oneembodiment, the composition includes, or consists essentially of, orconsists of, an extract of grape seed, an extract of guarana seed, anextract of prickly pear cactus, and an extract of turmeric root. In oneembodiment, the composition includes, or consists essentially of, orconsists of, an extract of guarana seed, an extract of red raspberryseed, an extract of prickly pear cactus and an extract of turmeric root.In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed, an extract of red raspberryseed, an extract of prickly pear cactus, and an extract of turmericroot.

In one embodiment, the composition includes, or consists essentially of,or consists of, an extract of grape seed, an extract of guarana seed, anextract of red raspberry seed, an extract of prickly pear cactus, and anextract of turmeric root.

In another aspect, the composition of the present invention isadministered at least once per day in combination with a dietary ornutritional supplement believed to have beneficial health effects. Forexample, Coenzyme Q10 (also known as CoQ10, Q10, vitamin Q10, ubiquinoneand ubidecarenone), a benzoquinone compound synthesized naturally by thehuman body, is used by cells of the body in oxidative metabolism or cellrespiration and as an endogenous antioxidant. An “antioxidant” is asubstance that protects cells from free radicals, which are highlyreactive chemicals often containing oxygen atoms, that are capable ofdamaging important cellular components, such as DNA and lipids. Theplasma level of CoQ10 has been used in studies as a measure of oxidativestress, a situation in which normal antioxidant levels are reduced.Various investigations have explored the usefulness of CoQ10 as atreatment for diseases, including, but not limited to, cancer andcardiovascular disease.

Idebenone, a synthetic analog of CoQ10, has been investigated in elderlypatients with dementia. Studies suggest that it may diminish nerve celldamage due to ischemia and facilitate memory and learning.

Huperzine A, a natural acetylcholinesterase inhibitor derived from theChinese herb Huperzia serrata, has antioxidant and neuroprotectiveproperties, and has been proposed as a disease-modifying treatment forAD.

Galantamine, an acetylcholinesterase inhibitor, is used to treatsymptoms of AD.

Vincamine and vinpocetine, a semisynthetic derivative of vincamine, analkaloid derived from the plant Vina minor L, are used in Europe, Japanand Mexico as pharmaceutical agents for the treatment of cerebrovascularand cognitive disorders.

Acetyl-L-carinitine, an acetylated derivative of carnitine, has beenshown to promote fatty acid beta-oxidation in liver and to prevent motornerve condition velocity slowing in diabetic rats.

Dehydroepiandrosterone (DHEA), a steroid, is being studied in theprevention of cancer. In the body, it is a precursor produced by theadrenal gland and converted to testosterone or the estrogens.

Phosphatidylcholine, a phospholipid that is a major component of cellmembranes, has putative activity as a cognition enhancer and incell-membrane repair.

Gingko, an herb, has putative properties as a neuroprotective agent, anantioxidant, a free-radical scavenger, a membrane stabilizer, and aninhibitor of platelet-activating factor. Sherpina, V. S., et al.,American Family Physician 68(5) 923-926 (2003). Gingko extract also hasbeen shown to inhibit beta-amyloid deposition. Id.

Ginseng, a Chinese herb, has been used for centuries in Asia as a curefor many maladies.

Research has shown that Vitamin E (DL-alpha-tocopherol), an essentialvitamin that functions as an antioxidant, can help preventcardiovascular disease and increase the immune response. It has beenhypothesized that Vitamin E and its analogs and derivatives may preventbrain cell damage by destroying toxic free radicals. The term “tocol”generally refers to 2-methyl-2-(4,8,12-trimetyltridecyl)chroman-6-ol;the term “tocopherol” generally refers to all mono, di, andtrimethyltocols, including, but not limited to, alpha-tocopherol(5,7,8-trimethyltocol), beta-tocopherol (5,8-dimethyltocol),gamma-tocopherol (7,8-dimethyltocol), delta-tocopherol (8-methyltocol),the term “tocotrienol” refers to2-methyl-2-(4,8,12-trimethyltrideca-3,7,11-trienyl)chroman-6-ol; and theterm “vitamin E” generally refers to all tocol and tocotrienolderivatives exhibiting qualitatively the biological activity ofalpha-tocopherol.

It is well-known that N-acetyl-cysteine (NAC) promotes cellularglutathione production, and thus reduces, or even prevents, oxidantmediated damage. Treatment with NAC provides beneficial effects in anumber of respiratory, cardiovascular, endocrine, infectious, and otherdisease settings.

B vitamins, such as folic acid, are known to reduce levels ofhomocysteine, an amino acid already linked, at high levels, to anincreased risk of heart attacks, strokes and Alzheimer's disease.

Lecithin, a lipid material composed of choline and inositol, is a majorcomponent of cell membranes. As used by producers of lecithin forcommercial use, the term “lecithin” refers to a complex mix ofphosphatides and other substances that contain phosphatidylcholine.

Choline (trimethyl ethanolamine), a quaternary saturated amineclassified as an essential nutrient by the Food and Nutrition Board ofthe Institute of Medicine, is a component of lecithin. Choline is neededby the body to make the neurotransmitter acetylcholine.

Fish oil, which is oil derived from the tissues of oily fish, naturallycontains the omega-3 fatty acids eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA). Some experts believe that fish oil can helpregulate cholesterol in the body. It also may help protect the brainfrom cognitive problems associated with Alzheimer's disease.

Deprenyl (selegiline, Eldepryl®), a monoamine oxidase inhibitor, isprescribed for the treatment of early-stage Parkinson's disease andsenile dementia.

The compositions of the invention can be used alone or in combinationwith other pharmaceuticals or herbals to prolong mental health, tomaintain or enhance cognitive functioning or memory, or to preservemental or physical well-being and health. The compositions can also beused to prevent or treat effects a number of ailments, including, butnot limited to, Alzheimer's disease; Parkinson's disease; heart disease;arthritis; age-related degeneration, functional impairments, anddiseases; diabetes, and cancer, have on cognitive function.

The effectiveness of the compositions and methods of the presentinvention can be assayed by a variety of protocols. The effects ofincreasing cognitive function in a human subject can be determined bymethods routine to those skilled in the art including, but not limitedto, both paper and pencil, and computer tests. One of skill in the artcan also directly measure PP2A methylation levels, tau proteinphosphorylation levels, neurofibrillary tangle formation andneurodegeneration in animal models.

Another aspect of the present invention provides a method of preparing abotanical extract that includes introducing a raw botanical to a solventhaving a dielectric constant ranging from about 2.3 to about 25 (e.g.,isopropyl alcohol, toluene and ethyl acetate) and allowing the mixtureto reside for a sufficient time (e.g., 4 hours, 8 hours, 16 hours, 24hours, 48 hours, or as necessary) at a set temperature (e.g., fromambient to boiling point of the corresponding solvent) to obtain a crudeextract. The method further includes filtering out remaining rawbotanical and evaporating the solvent from the crude extract underreduced pressure to obtain a dried crude extract (e.g., via a rotaryevaporator), and washing the dried crude extract with a polar solventhaving a dielectric constant of no less than 30 (e.g., water), heatingthe resulting mixture, and allowing to cool (e.g., allow to cool to ator approaching ambient). The method can further include collecting afiltrate from the cooled mixture mentioned above and washing thecollected filtrate with a non-polar solvent having a dielectric constantof no more than 2.0 (e.g., cyclohexane, hexane, heptane and isooctane).The method can further include filtering and drying the mixturementioned above to obtain the botanical extract.

The present invention further includes compositions that include,consist essentially of, or consists of an extract obtained by anyprocess described herein.

Another aspect of the present invention provides a method for inhibitingdemethylation of PP2A by PME-1 methylesterase comprising administeringto a subject in need thereof an effective amount of a botanical extract.Subjects that are in need of inhibition of demethylation of PP2A byPME-1 methylesterase, and hence can be administered the presentlydisclosed extracts or compositions, include, but are not limited to,subjects that exhibit, or are at risk for exhibiting, Tauhyper-phosphorylation, α-synculein hyper-phosphorylation, and/orabnormally elevated homocysteine levels. For example, the presentlydisclosed compositions can be administered to subjects who exhibitabnormal one carbon metabolism (e.g., subjects who have disruptions infolate, methionine and choline pathways).

According to another embodiment, the composition inhibits ademethylating activity of a demethylating enzyme that acts on a proteinphosphatase 2A enzyme and thereby stimulates methylation of the proteinphosphatase 2A enzyme. According to another embodiment, the compositioninhibits at least about 50% of the demethylating activity of thedemethylating enzyme. According to another embodiment, the demethylatingenzyme is a protein phosphatase 2A specific protein methylesterase.According to another embodiment, the demethylating activity of theprotein phosphatase 2A specific protein methylesterase is determined bymeasuring levels of protein phosphatase 2A methyl esterification.

In one embodiment, the subject in need of the presently disclosedextracts and compositions have been diagnosed with, or is at risk fordeveloping, Alzheimer' s Disease. In another embodiment, the subject inneed of the presently disclosed extracts and compositions have beendiagnosed with, or is at risk for developing, Parkinson's Disease.

In one embodiment, the subject is a healthy subject. For example, thehealthy subject may desire to prevent cognitive and/or motor functiondecline, or they may wish to improve upon their present cognitive andmotor function.

The invention also provides methods of enhancing memory in a human,which method includes administering a memory enhancing amount of apresently described composition (e.g., a pill, topical administration orcomestible). Methods of enhancing cognitive function in a human, themethod comprising the step of administering a cognitive functionenhancing amount of a presently described composition, wherein thecomposition inhibits at least 50% of the demethylating activity of thedemethylating enzyme as measured by levels of PP2A methylesterification.

According to the present invention, the compositions can be used inmethods of treating or preventing any disease, condition or disorderwhere defects in methylation metabolism appear to play a role asevidenced by an association of the disease, condition or disorder withplasma homocysteine levels that are elevated relative to normal plasmahomocysteine levels. Such diseases, conditions or disorders include, butare not limited to, neurodegenerative diseases, disorders or conditions,such as Parkinson's disease, neuropsychiatric diseases, disorders orconditions, such as bipolar disorder, Alzheimer's disease, heartdisease, arthritis, diabetes and certain cancers. The term“neurodegenerative” as used herein refers to a disease, condition ordisorder marked by the loss or diminution of an original nerve cellfunction, and the term “neuropsychiatric” relates to organic andfunctional diseases, conditions or disorders of the nervous system.

According to yet another embodiment, the compositions can be used inmethods of treating or preventing sleep disorders. Sleep disorders thatcan be treated using the compositions of the present invention include,but are not limited to, insomnia, narcolepsy, familial advancedsleep-phase syndrome (FASPS) and disruption to the circadian rhythm(e.g., jet lag).

The Clock (CLK) and Cycle (CYC) genes have been identified as positiveregulators of cycle, whereas transcription factors Period (PER) andTimeless (TIM) negatively regulate cycle, and are repressors of CKL andCYC. PER and TIM are high at the beginning of night, and nuclearlocalization in the middle of the night inhibits CYC/CLK transcriptionfactors, thus decreasing PER and TIM levels. In the morning hours,degradation of TIM and PER occurs, which promotes reactivation of PERand TIM expressions due to the release of the CYC/CLK/PER inhibitorycomplex. A new cycle starts through phosphorylation dependentdegradation of PER. PER, TIM and CLK are phosphorylated in a circadianfashion by Doubletime (casein kinase 1ε) which targets for degradationand inhibits nuclear import and casein kinase II, which is important fornuclear localization. Phosphorylation controls nuclear localization andstability. PP1/PP2A, mainly PP2A, antagonizes PER degradation. PP2A isimportant for PER stability and nuclear translocation. PP1 regulatesstability in a TIM dependent fashion.

PP2a dephosphorylates PER and TIM two main proteins in drosophila thatcontrol the circadian rhythm. Altering PP2A expression alters rhythmsalso affected by subunit expression (PER and TIM). See, Sathyanarayananet al. 2004 Post-translational modification of Drosophila PERIOD proteinby protein phosphatase 2A. Cell 116: 603. Human PERIOD2 (PER2)phosphorylated at Ser662 mutations at this site identified as familialadvanced sleep phase syndrome (FASPS).

Mutated PP2A does not result in sleep rebound (longer REM sleep aftersleep deprivation) which is instead associated with long term depressionin the hippocampus (statement made in 26 J. B. Calais etal./Neurobiology of Learning and Memory 122 (2015) 19-27); Norman etal., 2000; Long-term depression in the hippocampus in vivo is associatedwith protein phosphatase-dependent alterations in extracellularsignal-regulated kinase. Journal of Neurochemistry, 74(1), 192-198;Thiels et al., 2000 Protein phosphatase-mediated regulation of proteinkinase C during long-term depression in the adult hippocampus in vivo.Journal of Neuroscience, 20(19), 7199-7207. Twins (homologue of Bsubunit) and Widerborst (homologue of B′ subunit) are expressed in acircadian fashion; both which aid targeting of PER. PP2Adephosphorylates FREQUENCY in Neurospora. PP2A dephosphorylates andactivates white collar complex (transcription factor of the circadianclock in Neurospora).

According to another embodiment of the present invention, thecompositions can be used in methods of treating or preventing eye andvision disorders.

PP2A is implicated in visual transduction. PP2A translocation is lightdependent; PP2A with B56e dephosphorylates and co-elutes with phosducin(a phototransduction protein in the retina) this is not observed withBalpha or B′(B56a). Brown, B. et. al. Light driven Translocation of thePP2A complex regulates Light/Dark Dephosphorylation of Phosducin andRhodopsin. Biochemistry, 2002, 41(46);13526-13538.

In the retina PP2A dephosphorylates CaBP4, which is a neuronalCa(2+)-binding protein that is expressed in the retina and in thecochlea, and is essential for normal photoreceptor synaptic function.This action is light dependent. Haesseleer, F. et. Al. PP2Adephosphorylates CaBP4 and regulates CaBP4 function. Investi OphthalmolVis Sci. 2013, 54(2): 1214-1226. Mutations in CaBP4 are associated withcongenital stationary night blindness type 2B.

PP2A is also implicated in retina cell survival. Retinal-derived 661Wcells survival response through inhibition of PP2A and up regulation ofErk and Akt survival pathways. ROS production inhibits PP2A. IncreasedpTyr307 decreased methylation. This is also observed in retinal death inrd10 mice. Finnegan, S. et al. European Journal of Neuroscience. 2010;32(3): 322-334.

PP2A is also implicated in eye development. B56e required forIGF/PI3K/Akt pathway, which is important for eye induction and alsoregulates Hedgehog important for eye field separation (Xenopus).

According to yet another embodiment, the compositions of the presentinvention can be used in methods of treating or preventing a mentaldisorder. For example, the presently disclosed compositions can beadministered to subjects who exhibit abnormal one carbon metabolism(e.g., subjects who have disruptions in folate, methionine and cholinepathways). The term “mental disorder” refers to diseases characterizedas mood disorders, psychotic disorders, anxiety disorders, childhooddisorders, eating disorders, personality disorders, adjustment disorder,autistic disorder, delirium, dementia, multi-infarct dementia andTourette's disorder in the DSM-IV classification (Diagnosis andStatistical Manual of Mental Disorders, Fourth Edition, AmericanPsychiatric Association, Washington D.C., 1994). In one particularembodiment, the compositions of the present invention are used to treatan autistic disorder.

According to yet another embodiment, the present compositions can beused in methods of treating a traumatic brain injury (TBI).

According to yet another embodiment, the present compositions can beused in methods of treating or preventing a cardiovascular disease ordisorder. Cardiovascular diseases and disorders that can be treatedusing the compositions of the present invention include, but are notlimited to, ischemic heart disease, non-ischemic heart disease,myocardial infarction, tachy-pacing induced non-ischemic heart disease,heart failure, atherosclerosis, ischemic stroke, problems with heartvalves, and catecholaminergic-induced arrhythmia and symptoms thereof ina subject.

According to yet another embodiment, the present compositions can beused in methods of treating or preventing a metabolic disease ordisorder. Metabolic diseases and disorders that can be treated using thecompositions of the present invention include, but are not limited to,metabolic syndrome, insulin resistance, glucose intolerance,hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycysticovary syndrome (PCOS). and symptoms thereof in a subject.

According to yet another embodiment of the present invention, apharmaceutical preparation for promoting general health and well-beingin a mammalian subject, including a human, is provided that includes acognitive function-enhancing amount of an extract of the presentinvention and a pharmaceutically acceptable carrier.

In another embodiment, the subject exhibits, or is at risk forexhibiting, a skin disorder, medical condition or disease. In oneembodiment, the subject desires to maintain healthy skin and preventskin aging.

Another aspect of the present invention provides a method for inhibitinginflammation in a subject comprising administering to a subject aneffective amount of a botanical extract.

In certain embodiments, the present invention provides methods ofinhibiting inflammation, and uses of provided extracts and/orcompositions in the treatment of inflammation. In certain embodiments,the present invention provides uses of provided extracts and/orcompositions in the treatment of diseases that may benefit from edemainhibition, erythema inhibition and/or MPO inhibition, such as treatingor lessening the severity of inflammatory diseases or disordersincluding, but not limited to, inflammation (acute or chronic), asthma,autoimmune diseases, and chronic obstructive pulmonary disease (COPD)(e.g., emphysema, chronic bronchitis and small airways disease, etc.),inflammatory responses of the immune system, skin diseases (e.g.,reducing acute skin irritation for patients suffering from rosacea,atopic dermatitis, seborrheic dermatitis, psoriasis), irritable bowelsyndrome (e.g., Crohn's disease and ulcerative colitis, etc.), andcentral nervous system disorders (e.g., Parkinson's disease).

The present invention also relates to treating or lessening the severityof one or more diseases in which protein inhibitors that modulate theG-protein signaling cascade are known to play a role. Specifically, thepresent invention relates to a method of treating or lessening theseverity of inflammatory diseases or disorders selected frominflammation (acute or chronic), inflammatory diseases or disorders(e.g., asthma, autoimmune diseases, and COPD including emphysema,chronic bronchitis and small airways disease, etc.), inflammatoryresponses of the immune system, skin diseases (e.g., reducing acute skinirritation for patients suffering from rosacea, atopic dermatitis,seborrheic dermatitis, psoriasis), irritable bowel syndrome (e.g.,Crohn's disease and ulcerative colitis, etc.), and Parkinson's disease,wherein the method comprises administering to a patient in need thereofa composition of the present invention.

In certain embodiments, the provided extracts of the present inventionare capable of effectively inhibiting inflammatory responses that aremediated by G-proteins or GPCRs in neutrophils, macrophages andplatelets. Thus, provided extracts are inhibitors of edema, erythema andmyeloperoxidase and are therefore useful for treating one or moredisorders associated with inflammatory diseases or disorders asdescribed herein. In particular, the present invention encompasses thefinding that certain extracts having superior in vivo activity thanother extracts in the same class. Therefore, such extracts areadministered to a subject suffering from or susceptible to one or moreinflammatory diseases or disorders.

In certain embodiments, the treatment of inflammatory diseases ordisorders is achieved using extracts without having the side effects ofcorticosteroids or NSAIDS.

In certain embodiments, such extracts are administered in vitro. Incertain embodiments such extracts are administered in vivo.

Another aspect of the present invention is directed to methods oftreating, preventing, or ameliorating inflammation by administering aneffective amount of a provided extract.

In some embodiments, one or more inventive extracts, alone or togetherwith one or more other pharmaceutically active agents, is used to whitenskin. In some such embodiments, the extract is applied topically.

In general, the actual quantity of provided extracts of the inventionadministered to a patient will vary depending on the severity and typeof indication, the mode of administration, the particular extract used,the formulation used, and the response desired.

The dosage for treatment is administration, by any of the foregoingmeans or any other means known in the art, of an amount sufficient tobring about the desired therapeutic effect. Thus, an effective amountincludes an amount of a provided extract (or mixture of providedextracts) or pharmaceutical composition of this invention that issufficient to induce a desired effect, including specifically ananti-inflammation effect.

In general, the provided extracts of the present invention are highlyactive. For example, a provided extract can be administered at about 10μg/kg to about 50 mg/kg body weight, depending on the specific providedextract selected, the desired therapeutic response, the route ofadministration, the formulation and other factors known to those ofskill in the art.

In one embodiment, the botanical extract to be administered to subjectsas described herein, is obtained from at least one fruit source selectedfrom the group consisting of: juniper berry, schisandra, and strawberry.In another embodiment, the botanical extract is obtained from at leastone seed source (e.g., avocado, black raspberry, blueberry, celery,cranberry, fennel, grape, guarana and red raspberry). In one particularembodiment, the seed source is selected from grape, guarana and redraspberry.

In another embodiment, the botanical extract is obtained from a leastone root, bark or leaf source (e.g., maca root, goldenseal root,turmeric root, magnolia bark, pygeum bark, red raspberry leaf). In astill further embodiment, the root, bark or leaf source is turmericroot.

In another embodiment, the botanical extract is obtained from at leastone natural source selected from the group consisting of: almond, cocoapowder, Echinacea angustifolia, prickly pear cactus and walnut. In oneparticular embodiment, the natural source is prickly pear cactus.

The compositions of the present invention may be in a form suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, solutions, dispersible powders or granules, emulsions, hardor soft capsules, syrups or elixirs, pastes, gels or the like.Compositions intended for oral use may be prepared according to anyknown method, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to providepharmaceutically elegant and palatable compositions. Tablets may containthe active ingredient(s) in admixture with non-toxicpharmaceutically-acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed. They alsomay be coated for controlled delivery. For example, a “delayed release”dosage form releases a product or substance at a time other thanpromptly after administration. Examples of delayed-release systemsinclude repeat-action tablets and capsules, and enteric-coated tabletswhere timed release is achieved by a barrier coating.

Compositions of the present invention also may be formulated for oraluse as hard gelatin capsules, where the active ingredient(s) is(are)mixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or soft gelatin capsules wherein the activeingredient(s) is (are) mixed with water or an oil medium, for example,peanut oil, liquid paraffin, or olive oil.

The compositions of the present invention may be formulated as aqueoussuspensions wherein the active ingredient(s) is (are) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatidesuch as lecithin, or condensation products of an alkylene oxide withfatty acids, for example, polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample, heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions also may contain one or more coloring agents,one or more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Compositions of the present invention may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil, forexample arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil, such as liquid paraffin. The oily suspensions may contain athickening agent, for example, beeswax, hard paraffin or cetyl alcohol.Sweetening agents, such as those set forth above, and flavoring agentsmay be added to provide a palatable oral composition. These compositionsmay be preserved by the addition of an antioxidant such as ascorbicacid.

Compositions of the present invention may be formulated in the form ofdispersible powders and granules suitable for composition of an aqueoussuspension by the addition of water. The active ingredient in suchpowders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients, or example,sweetening, flavoring and coloring agents also may be present.

The compositions of the invention also may be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample, olive oil or arachis oil, or a mineral oil, for example aliquid paraffin, or a mixture thereof. Suitable emulsifying agents maybe naturally-occurring gums, for example, gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate. The emulsions also may contain sweetening andflavoring agents.

The compositions of the invention also may be formulated as syrups andelixirs. Syrups and elixirs may be formulated with sweetening agents,for example, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations also may contain a demulcent, a preservative, and flavoringand coloring agents. Demulcents are protective agents employed primarilyto alleviate irritation, particularly mucous membranes or abradedtissues. A number of chemical substances possess demulcent properties.These substances include the alginates, mucilages, gums, dextrins,starches, certain sugars, and polymeric polyhydric glycols. Othersinclude acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,propylene glycol, sodium alginate, tragacanth, hydrogels and the like.

Liquid based oral dosage forms, like their solid counterparts, can, incertain embodiments contain at least 0.1 mg of a provided extract. Oneskilled in the art will be able to properly formulate a liquidformulation containing an appropriate amount of a provided extract perfluidic ounce, depending on the additive or carrier selected.

Formulations suitable for buccal administration include tablets andlozenges comprising an extract in a flavored base, such as sucrose,acacia or tragacanth; and pastilles comprising the extract in an inertbase, such as gelatin and glycerin or sucrose and acacia.

For topical administration formulations, any of a variety of creams,ointments, gels, cleansers, lotions and the like may be employed. Formost pharmaceutical formulations, non-active ingredients will constitutethe greater part, by weight or volume, of the preparation. Forpharmaceutical formulations, it is also contemplated that any of avariety of measured-release, slow-release or time-release formulationsand additives may be employed, so that the dosage may be formulated soas to effect delivery of a provided extract over a period of time. Forexample, gelatin, sodium carboxymethylcellulose and/or other cellulosicexcipients may be included to provide time-release or slower-releaseformulations, especially for administration by subcutaneous andintramuscular injection. Other additives which may be used includevaseline, lanoline, polyethylene glycols, alcohols, transdermalenhancers, and combinations of two or more thereof.

In some embodiments, formulations suitable for topical applicationachieve transdermal delivery. Transdermal pharmaceutical devices includepatches, occlusive dressings, occlusive formulations, hypodermic sprays,iontophoretic systems, gels and infusion pumps, all of which are wellknown in the art. A transdermal patch which includes a pharmaceuticalmay generally include a backing layer impermeable to the pharmaceutical,a reservoir to house the pharmaceutical, and an adhesive cover to beremoved upon use of the patch and for adhesion to the skin of a patient.

Formulations suitable for transdermal administration may also bepresented as medicated bandages or discrete patches adapted to remain inintimate contact with the epidermis of the recipient for a prolongedperiod of time. Representative examples of suitable transdermal patchesinclude, for example, those developed by NeuroDerm Ltd (Israel) and/orthat used to deliver estradiol, for example, those developed by NovogynePharmaceuticals. Formulations suitable for transdermal administrationmay also be delivered by iontophoresis (passage of a small electriccurrent (^(˜)15 mA) to “inject” electrically charged ions into the skin)through the skin. For this, the dosage form typically takes the form ofan optionally buffered aqueous solution of the active extract.

In practical use, a provided extract of the present invention can becombined as the active ingredient in an admixture with a pharmaceuticalcarrier according to conventional pharmaceutical compounding techniques.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, for example, oral, parenteral(including intravenous), urethral, vaginal, nasal, dermal, transdermal,pulmonary, deep lung, inhalation, buccal, sublingual, or the like. Inpreparing the compositions for oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations, such as,for example, suspensions, elixirs and solutions; or carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like in thecase of oral solid preparations such as, for example, powders, hard andsoft capsules and tablets.

According to yet another embodiment of the present invention, acomestible for promoting general health and well-being in a mammaliansubject, including a human, including one or more of the extractsdescribed herein. According to another embodiment, the comestible is abeverage. According to another embodiment, the beverage is selected fromthe group consisting of a drink comprising water, a fruit drink, acoffee, a tea, an energy drink, a baby formula, an adult nutritionaldrink, a health drink, and a sports drink. According to anotherembodiment, the comestible is a food. According to another embodiment,the comestible is a cereal. According to another embodiment, thecomestible is a chewing gum. According to another embodiment, thecomestible is a candy.

EXAMPLES Example 1 Preparation of Extracts

Various water-soluble oligosaccharides and starch in botanicals canreduce the specific activity of the extract, making the extract prone tobacterial contamination and growth; and change its appearance to stickythick oil or to glassy caramel-looking substance. These properties makemost of aqueous extracts difficult in manufacture (drying, emptying fromreactor, drumming, etc.) and affect products shelf life and overallsafety. Furthermore, removing various naturally occurring in a givenbotanic extract non-biologically active oils and lipids result in finalextract to be a free flowing powder improving ease of manufacturing andblending into a product. Therefore, a process for manufacturing theseextracts was designed to take these factors into account.

A raw botanical (10 g) was shaken with isopropyl alcohol (100 mL) in aflask at 65° C. for 16 hrs. The resulting isopropanolic extract wasfiltered and concentrated to dryness on a rotavap. The crude extract washeated with water (100 ml) to remove caffeine and oligosaccharides,cooled to room temperature and collected by filtration. The solid waswashed with hexane to remove oils and lipids to yield upon filtering anddrying the final extract. The yields are listed in the Table 1.

TABLE 1 Extract's yields Fruit Extract Yield (%) Appearance Almond 4 Offwhite Avocado 18 Yellow semi-solid Avocado Seed 2 Gold oil BlackRaspberry 2 Reddish gold Blueberry 1 Pale yellow Brazil Nut 14 Clear oilBromalin <1 Off white solid Celery 3 Clear Cocoa Butter <1 Clear/Whiteoil Cocoa Powder <1 Off white solid Coconut <1 White solid Coffee 9Brown solid Cranberry 2 Pale yellow Echinacea Angustifolia 11 ClearFennel Seed 2 Pale gold oil Goldenseal Root 4 Brownish yellow Grape Seed8 Reddish-brown solid Grape Fruit (Oil) <1 Clear Guarana Seed 21 Tansolid Hazelnut 4 Clear oil Juniper Berry 7 Yellow Maca Root 7 BrownMagnolia Bark 11 Carmel Maqui Berry 18 Off-white Mulberry 7 Off-whitePomegranate <1 Red sticky solid Pygeum Bark 11 Off white Prickly PearCactus 14 Dark green solid Prickly Pear Fruit 8 Off white solid RedRaspberry 7 Red solid Red Raspberry Leaf 3 Tan Schisandra <1 TanStrawberry 2 Pink Walnut 16 Golden oil

Example 2 Natural Extracts Effect on PP2A Methylation

Extracts, as shown in Tables 1-4 below, were created using isopropanolor toluene-based extraction methods at 65° C. as generally described inExample 1. These extracts were assayed for their ability to preserve themethylation status of PP2A AC dimer in the presence of proteinphosphatase methylesterase-1 (PME-1), using a radioactive filter bindingassay. Methylated PP2A was made by incubating PP2A, protein phosphatasemethyl transferase (PPMT) and [3H]-SAM for 1 hr. PME-1 was incubatedwith varying concentrations of botanical extracts prior to the additionof 50 nM of methylated PP2A. The reaction was allowed to continue for 30minutes at room temperature. 10% TCA was added to stop the reaction andincubated for 1 hour. Remaining buffer was filtered and total countsremaining on the filter plate were measured using a scintillationcounter. IC₅₀ values were generated from dose-response curves using afour-parameter logistic curve fit (SigmaPlot).

The following results were obtained:

TABLE 1 Fruit extracts Estimated IC₅₀ Fruit Extract (μg/mL) Avocado >25Blueberry >50 Bromalin >50 Cranberry >30 Coconut >27 Grape Fruit (Oil)25 Juniper Berry 5 Maqui Berry >10 Mulberry >10 Pomegranate (Juice) >27Prickly Pear Fruit 15.6 Schisandra 6 Strawberry >5

TABLE 2 Seed extracts Estimated IC₅₀ Seed Extract (μg/mL) Avocado 6Black Raspberry 6 Blueberry 8 Celery 7 Coffee 5 Cranberry 10 Fennel 1Grape 0.4 Guarana 1.5 Red Raspberry 1

TABLE 3 Root, bark & leaf extracts Estimated IC₅₀ Natural Extract(μg/mL) Goldenseal Root 4 Maca Root 3 Magnolia Bark 2 Ppygum Bark 3 RedRaspberry Leaf 2 Tumeric Root 2.9

TABLE 4 Other natural extracts Estimated IC₅₀ Natural Extract (μg/mL)Almond >5 Hazelnut >10 Cocoa Butter 12.5 Cocoa Powder 6.25 EchinaceaAngustifolia 4 Prickly Pear Cactus 52.5 Walnut 6

Only a subset of the botanical extracts tested demonstrated preventionof PP2A demethylation. Results indicate a distinction between extractcategories, where the seed and root, bark and leaf categoriesconsistently prevented PP2A demethylation with an IC₅₀ better than 5μg/mL.

Example 3 Natural Extracts Effect on ROS Scavenging (Antioxidant)

Botanical extracts ability to prevent oxidation of ABTS by metmyoglobinwas analyzed. The antioxidant capacity of select botanical extracts wasevaluated in vitro using the ABTS (2,2′-Azino-bis-[3-ehthylbenzthiazoline sulphonate]) antioxidant kit(Cayman Chemical Company; Ann Arbor, Mich.). Botanical extracts wereincubated with both metmyoglobin and chomogen, ABTS. The reaction wasinitiated upon addition of hydrogen peroxide and the oxidation of theABTS to ABTS^(•+) was monitored via absorbance at 750 nm. EC₅₀ valueswere generated from dose-response curves using a four-parameter logisticcurve fit (SigmaPlot), and are shown below in Table 5.

Example 4 Natural Extracts Effect on LPS-Induced Cytokine Release inPBMCs (Anti-Inflammatory)

Botanical extracts ability to inhibit the production of inflammatorycytokines TNF-α and IL-8 in PBMCs when challenged withlipopolysaccharides (LPS) through the administration of select botanicalextracts was analyzed.

Normal human peripheral blood mononuclear cells (PBMCs) were obtainedfrom a single donor, purchased from Zenbio (Research Triangle Park, NC).Cells plated at a seeding density of 0.33×10⁶ cells/well. A two-hourpre-incubated period was performed with 150 μg/mL botanical extractsonly fresh media in triplicates. Cells were then stimulated with 5 μg/mLLPS for 18 hours in the presence or absence of extract. Afterincubations, media supernatants were collected and analyzed by sandwichELISA following the manufacturers protocols. Results are shown below inTable 5.

Prevention of +LPS Inhibition of ABTS TNF-a (pg/mL) PP2A Oxidation 150μg/mL Extract Demethylation (Antioxidant) Average ± Extract Solvent IC₅₀(μg/mL) EC₅₀ (μg/mL) SEM % Inhibition Fennel Seed Water >25 >500   78 ±3.9 56.2 Isopropanol 6 >500 80.5 ± 9.2 62.5 Toluene 1 >500 68.9 ± 19 64.5 Grape Seed Water 0.8 6.55 ± 0.8  147 ± 6.4 22.9 Isopropanol 0.46.14 ± 2.6 Cytotoxic Cytotoxic Toluene >12.5 >500 118.2 ± 15   28.8Guarana Seed Water 8.7 23.2 58.5 ± 2.4 76.4 Isopropanol 1.3 7.83   75 ±2.8 69.5 Toluene 1.1 67.7 Cytotoxic Cytotoxic Prickly Pear Isopropanol52.5 163 74.9 ± 4.3 59.1 Cactus Toluene 4.5 174   143 ± 30.6 −2.8Prickly Pear Water >50 >500 53.3 ± 1.6 79.8 Fruit Isopropanol 15.6 >500  192 ± 36.9 −9.5 Toluene 6.2 >500 97.9 ± 0.5 51 Red Water >110 34.9 100 ± 5.3 45.6 Raspberry Seed Isopropanol 1 8.01 ± 0.4 59.5 ± 2.1 84Toluene 1 >500 N.D. N.D. Turmeric Root Water >50 >500  51.1 ± 0.75 80.7Isopropanol 2.9 11.4 36.8 ± 5.1 75 Toluene 29 11.2 68.2 ± 6.5 65.1

All publications, patent and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade therein without departing from the scope of the inventionencompassed by the appended numbered embodiments. Further, allembodiments included herein are given solely for the purpose ofillustration and are not to be construed as limitations of the presentinvention, as many variations thereof are possible without departingfrom the spirit and scope of the invention.

1. A composition for use in for inhibiting demethylation of PP2A byPME-1 methylesterase comprising an extract of one or more botanicalsselected from the group consisting of juniper berry fruit, schisandrafruit, strawberry fruit, avocado seeds, black raspberry seeds, blueberryseeds, celery seeds, cranberry seeds, fennel seeds, guarana seeds, redraspberry seeds, maca root, goldenseal root, turmeric root, magnoliabark, pygeum bark, red raspberry leaf, almond, cocoa powder, Echinaceaangustifolia, prickly pear cactus and walnut.
 2. The composition ofclaim 1, wherein the one or more botanicals is selected from the groupconsisting of guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 3. The composition of any one of claim 1 or 2, whereinthe composition further includes an extract of grape seed.
 4. Thecomposition of claim 1, wherein the extract is obtained from a solventselected from solvents having a dielectric constant ranging from about2.3 to about
 25. 5. The composition of claim 4, wherein the extract isobtained from one or more of isopropyl alcohol, toluene, and ethylacetate.
 6. The composition of claim 4, wherein the extract issubstantially free of polar components soluble in solvents with adielectric constant no less than
 30. 7. The composition of claim 6,wherein the solvent with a dielectric constant no less than 30 is water.8. The composition of claim 6, wherein the extract is substantially freeof non-polar components soluble in solvents with a dielectric constantno more than 2.0.
 9. The composition of claim 8, wherein the solventwith a dielectric constant no more than 2.0 is selected fromcyclohexane, hexane, heptane or isooctane.
 10. A composition for use infor inhibiting the formation of free radicals and reactive oxygenspecies comprising an extract of one or more botanicals selected fromthe group consisting of grape seed, guarana seed, red raspberry seed,prickly pear cactus, and turmeric root.
 11. The composition of claim 10wherein the extract is obtained from a solvent selected from solventshaving a dielectric constant ranging from about 2.3 to about
 25. 12. Thecomposition of claim 11, wherein the extract is obtained from one ormore of isopropyl alcohol, toluene, and ethyl acetate.
 13. Thecomposition of claim 11, wherein the extract is substantially free ofpolar components soluble in solvents with a dielectric constant no lessthan
 30. 14. The composition of claim 13, wherein the solvent with adielectric constant no less than 30 is water.
 15. The composition ofclaim 11, wherein the extract is substantially free of non-polarcomponents soluble in solvents with a dielectric constant no more than2.0.
 16. The composition of claim 15, wherein the solvent with adielectric constant no more than 2.0 is selected from cyclohexane,hexane, heptane or isooctane.
 17. A composition for use in forinhibiting inflammation comprising an extract of one or more botanicalsselected from the group consisting of grape seed, guarana seed, redraspberry seed, prickly pear cactus, and turmeric root.
 18. Thecomposition of claim 17 wherein the extract is obtained from a solventselected from solvents with a dielectric constant ranging from about 2.3to about
 25. 19. The composition of claim 18, wherein the extract isobtained from one or more of isopropyl alcohol, toluene, and ethylacetate.
 20. The composition of claim 18, wherein the extract issubstantially free of polar components soluble in solvents with adielectric constant no less than
 30. 21. The composition of claim 20,wherein the solvent with a dielectric constant no less than 30 is water.22. The composition of claim 20, wherein the extract is substantiallyfree of non-polar components soluble in solvents with a dielectricconstant no more than 2.0.
 23. The composition of claim 22, wherein thesolvent with a dielectric constant no more than 2.0 is selected fromcyclohexane, hexane, heptane or isooctane.
 24. A composition for use infor inhibiting demethylation of PP2A by PME-1 methylesterase, inhibitingthe formation of free radicals and reactive oxygen species, andinhibiting inflammation comprising an extract of one or more botanicalsselected from the group consisting of grape seed, guarana seed, redraspberry seed, prickly pear cactus, and turmeric root.
 25. Thecomposition of claim 24, wherein the composition comprises comprising anextract of two or more botanicals selected from the group consisting ofgrape seed, guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 26. The composition of claim 25, wherein the compositioncomprises comprising an extract of three or more botanicals selectedfrom the group consisting of grape seed, guarana seed, red raspberryseed, prickly pear cactus, and turmeric root.
 27. The composition ofclaim 26, wherein the composition comprises comprising an extract offour or more botanicals selected from the group consisting of grapeseed, guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 28. The composition of claim 27, wherein the compositioncomprises an extract of grape seed, an extract of guarana seed, anextract of red raspberry seed, an extract of prickly pear cactus, and anextract of turmeric root.
 29. A method of preparing a botanical extractcomprising: (a) introducing a raw botanical to a solvent having adielectric constant ranging from about 2.3 to about 25 and allowing themixture to reside for a sufficient time to obtain a crude extract; (b)filtering out remaining raw botanical and evaporating the solvent fromthe crude extract under reduced pressure to obtain a dried crudeextract; (c) washing the dried crude extract with a polar solvent havinga dielectric constant of no less than 30, heating the resulting mixture,and allowing to cool to ambient; (d) collecting a filtrate from thecooled mixture from step (c); and (e) washing the collected filtratewith a non-polar solvent having a dielectric constant of no more than2.0; and (f) filtering and drying the mixture from step (e) to obtainthe botanical extract.
 30. The method of claim 29, wherein the solventhaving a dielectric constant ranging from about 2.3 to about 25 isselected from isopropyl alcohol, toluene and ethyl acetate.
 31. Themethod of claim 29, wherein the solvent having a dielectric constantranging from about 2.3 to about 25 is evaporated from the crude extractby a rotary evaporator.
 32. The method of claim 29, wherein the polarsolvent having a dielectric constant of no less than 30 is water. 33.The method of claim 29, wherein the non-polar solvent having adielectric constant of no more than 2.0 is selected from cyclohexane,hexane, heptane and isooctane.
 34. A composition comprising an extractprepared by the process of any one of claims 29-33.
 35. A method forinhibiting demethylation of PP2A by PME-1 methylesterase comprisingadministering to a subject in need thereof an effective amount of abotanical extract.
 36. The method of claim 35, wherein the subjectexhibits, or is at risk for exhibiting, Tau hyper-phosphorylation. 37.The method of claim 35, wherein the subject exhibits, or is at risk forexhibiting, α-synculein hyper-phosphorylation.
 38. The method of claim35, wherein the subject exhibits, or is at risk for exhibiting,abnormally elevated homocysteine levels.
 39. The method of claim 35,wherein the subject has been diagnosed with, or is at risk fordeveloping, Alzheimer's Disease.
 40. The method of claim 35, wherein thesubject has been diagnosed with, or is at risk for developing,Parkinson's Disease.
 41. The method of claim 35, wherein the subject isa healthy subject.
 42. The method of any one of claims 35-41, whereinthe subject desires to prevent cognitive and/or motor function decline.43. The method of claim 35, wherein the subject exhibits, or is at riskfor exhibiting, a skin disorder, medical condition or disease.
 44. Themethod of any one of claims 35, wherein the subject desires to maintainhealthy skin and prevent skin aging.
 45. The method of claim 35, whereinthe botanical extract is obtained from at least one fruit sourceselected from the group consisting of: juniper berry, schisandra, andstrawberry.
 46. The method of claim 35, wherein the botanical extract isobtained from at least one seed source.
 47. The method of claim 46,wherein the seed source is selected from the group consisting of:avocado, black raspberry, blueberry, celery, cranberry, fennel, grape,guarana and red raspberry.
 48. The method of claim 47, wherein the seedsource is selected from grape, guarana and red raspberry.
 49. The methodof claim 35, wherein the botanical extract is obtained from a least oneroot, bark or leaf source.
 50. The method of claim 49, wherein the root,bark or leaf source is selected from the group consisting of maca root,goldenseal root, turmeric root, magnolia bark, pygeum bark, redraspberry leaf.
 51. The method of claim 50, wherein the root, bark orleaf source is turmeric root.
 52. The method of claim 35, wherein thebotanical extract is obtained from at least one natural source selectedfrom the group consisting of: almond, cocoa powder, Echinaceaangustifolia, prickly pear cactus and walnut.
 53. The method of claim52, wherein the natural source is prickly pear cactus.
 54. The method ofclaim 35, wherein the botanical extract is a botanical extract obtainedby the method of any one of claims 29-33.
 55. The method of any one ofclaims 35-54, wherein the botanical extract is topically administered asa cream, lotion, cleanser, ointment or other pharmaceutically acceptabletopical dosage form.
 56. The method of any one of claims 35-54, whereinthe botanical extract is orally administered as a pill, tablet, capsule,syrup or a drink or other pharmaceutically acceptable oral dosage form.57. The method of any one of claims 35-54, wherein the botanical extractis administered together with other botanicals and vitamins.
 58. Amethod for treating or preventing a sleep disorder comprisingadministering to a subject in need thereof an effective amount of abotanical extract.
 59. The method of claim 58, wherein the sleepdisorder is selected from insomnia, narcolepsy, familial advancedsleep-phase syndrome (FASPS) and disruption to the circadian rhythm. 60.The method of claim 58, wherein the botanical extract is selected fromone or more of grape seed, juniper berry fruit, schisandra fruit,strawberry fruit, avocado seeds, black raspberry seeds, blueberry seeds,celery seeds, cranberry seeds, fennel seeds, guarana seeds, redraspberry seeds, maca root, goldenseal root, turmeric root, magnoliabark, pygeum bark, red raspberry leaf, almond, cocoa powder, Echinaceaangustifolia, prickly pear cactus and walnut.
 61. The method of claim60, wherein the botanical extract is selected from grape seed, guaranaseed, red raspberry seed, prickly pear cactus, and turmeric root.
 62. Amethod for treating or preventing an eye or vision disorder comprisingadministering to a subject in need thereof an effective amount of abotanical extract.
 63. The method of claim 62, wherein the botanicalextract is selected from one or more of grape seed, juniper berry fruit,schisandra fruit, strawberry fruit, avocado seeds, black raspberryseeds, blueberry seeds, celery seeds, cranberry seeds, fennel seeds,guarana seeds, red raspberry seeds, maca root, goldenseal root, turmericroot, magnolia bark, pygeum bark, red raspberry leaf, almond, cocoapowder, Echinacea angustifolia, prickly pear cactus and walnut.
 64. Themethod of claim 63, wherein the botanical extract is selected from grapeseed, guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 65. A method for treating or preventing a metabolicdisease or disorder comprising administering to a subject in needthereof an effective amount of a botanical extract.
 66. The method ofclaim 65, wherein the metabolic disease or disorder is selected frommetabolic syndrome, insulin resistance, glucose intolerance,hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycysticovary syndrome (PCOS).
 67. The method of claim 65, wherein the botanicalextract is selected from one or more of grape seed, juniper berry fruit,schisandra fruit, strawberry fruit, avocado seeds, black raspberryseeds, blueberry seeds, celery seeds, cranberry seeds, fennel seeds,guarana seeds, red raspberry seeds, maca root, goldenseal root, turmericroot, magnolia bark, pygeum bark, red raspberry leaf, almond, cocoapowder, Echinacea angustifolia, prickly pear cactus and walnut.
 68. Themethod of claim 67, wherein the botanical extract is selected from grapeseed, guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 69. A method for treating or preventing a mental disordercomprising administering to a subject in need thereof an effectiveamount of a botanical extract.
 70. The method of claim 69, wherein themental disorder is selected from a mood disorder, a psychotic disorder,an anxiety disorder, a childhood disorder, an eating disorder, apersonality disorder, an adjustment disorder, an autistic disorder,delirium, dementia, multi-infarct dementia and a Tourette's disorder.71. The method of claim 70, wherein the mental disorder is an autisticdisorder.
 72. The method of claim 69, wherein the botanical extract isselected from one or more of grape seed, juniper berry fruit, schisandrafruit, strawberry fruit, avocado seeds, black raspberry seeds, blueberryseeds, celery seeds, cranberry seeds, fennel seeds, guarana seeds, redraspberry seeds, maca root, goldenseal root, turmeric root, magnoliabark, pygeum bark, red raspberry leaf, almond, cocoa powder, Echinaceaangustifolia, prickly pear cactus and walnut.
 73. The method of claim72, wherein the botanical extract is selected from grape seed, guaranaseed, red raspberry seed, prickly pear cactus, and turmeric root.
 74. Amethod for treating a traumatic brain injury comprising administering toa subject in need thereof an effective amount of a botanical extract.75. The method of claim 74, wherein the botanical extract is selectedfrom one or more of grape seed, juniper berry fruit, schisandra fruit,strawberry fruit, avocado seeds, black raspberry seeds, blueberry seeds,celery seeds, cranberry seeds, fennel seeds, guarana seeds, redraspberry seeds, maca root, goldenseal root, turmeric root, magnoliabark, pygeum bark, red raspberry leaf, almond, cocoa powder, Echinaceaangustifolia, prickly pear cactus and walnut.
 76. The method of claim75, wherein the botanical extract is selected from grape seed, guaranaseed, red raspberry seed, prickly pear cactus, and turmeric root.
 77. Amethod for inhibiting inflammation in a subject comprising administeringto a subject an effective amount of a botanical extract obtained from atleast one source selected from the group consisting of grape seed,guarana seed, red raspberry seed, prickly pear cactus and turmeric root.78. The method of claim 77 wherein the botanical extract is a botanicalextract obtained by the process of any one of claims 29-33.
 79. Themethod of any one of claims 77-78, wherein the botanical extract istopically administered as a cream, lotion, cleanser, ointment or anygenerally accepted topical dosage form.
 80. The method of any one ofclaims 77-78 wherein the botanical extract is orally administered as apill, tablet, capsule, syrup or a drink or other pharmaceuticallyacceptable oral dosage form.
 81. The method of any one of claims 77-78,wherein the botanical extract is administered together with otherbotanicals and vitamins.
 82. A method for inhibiting demethylation ofPP2A by PME-1 methylesterase, inhibiting the formation of free radicalsand reactive oxygen species, and inhibiting inflammation in a subjectcomprising administering to a subject in need thereof a compositioncomprising one or more botanical extracts obtained from at least onesource selected from the group consisting of grape seed, guarana seed,red raspberry seed, prickly pear cactus and turmeric root.
 83. Themethod of claim 82, wherein the composition comprises comprising anextract of two or more botanicals selected from the group consisting ofgrape seed, guarana seed, red raspberry seed, prickly pear cactus, andturmeric root.
 84. The method of claim 83, wherein the compositioncomprises comprising an extract of three or more botanicals selectedfrom the group consisting of grape seed, guarana seed, red raspberryseed, prickly pear cactus, and turmeric root.
 85. The method of claim84, wherein the composition comprises comprising an extract of four ormore botanicals selected from the group consisting of grape seed,guarana seed, red raspberry seed, prickly pear cactus, and turmericroot.
 86. The method of claim 85, wherein the composition comprises anextract of grape seed, an extract of guarana seed, an extract of redraspberry seed, an extract of prickly pear cactus, and an extract ofturmeric root.
 87. The method of any of claims 82-86, wherein saidextracts are administered in a single composition.
 88. The method ofclaim 87, wherein said extracts are orally administered.
 89. The methodof claim 87, wherein said extracts are topically administered.